DESCRIPTION: Cardiovascular disease remains the #1 killer in the developed world. Loss of cardiomyocytes due to myocardial infarction or chronic apoptosis can lead to heart failure, affecting more than 23 million people worldwide. We currently lack a nuanced understanding of how the mechanisms of dysfunction of heart disease are linked to contractile phenotypes at the protein, sarcomere and cellular levels. Unfortunately, the scarcity of human heart tissue and the inability to maintain mature, primary cardiomyocytes in vitro has hindered investigation of the basic mechanisms of cardiotoxicity and human heart disease and physiology of cardiomyocytes. Conversely, human induced pluripotent stem cell-derived cardiomyocytes are readily available (even commercially), can be maintained for months in culture, and frozen for future use. A crucial motivation for this work is the divergence between human cardiomyocytes and animal model cardiomyocytes in physiology, structural composition, and fundamental biology. These differences are particularly acute when screening cardiotoxicity of drugs or treatments for use in humans. Human induced pluripotent stem cell-derived cardiomyocytes seem to hold great promise in this regard, but current protocols for deriving cells yield heterogeneous populations in terms of structure, function, contractility, and other crucial parameters. In this application, we seek to establish a high-risk, high-reward paradigm shift: that quantitative analysis of myofibril organization in engineered single cardiomyocytes with physiological shape and sarcomere organization will empower researchers to overcome the heterogeneities observed in human induced pluripotent stem cell-derived cardiomyocyte populations, positioning the contractile behavior and myofibril organization of human induced pluripotent stem cell-derived cardiomyocyte as models of cardiotoxicity and diseases of the myocardium (cardiomyopathies). We will engineer the morphology and subcellular myofibril alignment of human induced pluripotent stem cell-derived cardiomyocytes through their interface with mechanically tuned cell culture environments. By providing in situ non-destructive functional assessment, while driving maturity in single iPSC-cardiomyocytes, we will enable quantitative studies of contractility, work and power in terminally differentiated cardiomyocytes. These models of matured stem cell-derived cardiomyocytes also have the potential to avoid the problems of poor long-term survival of primary cardiomyocyte models in vitro, to reduce our reliance on animal models and to avoid their known differences from human cells. Our proposed project provides methods and systems for sustaining stem cell-derived cardiomyocytes in biomimetic culture conditions along with non-destructive contractility assays required to assess the function of these cells before and after interventions to rescue healthy phenotypes. We further aim to deploy these model systems and methods to characterize the biophysics of mutations causing heritable cardiomyopathies. We seek to demonstrate models suitable for future translation towards high- throughput testing of therapies with patient specificity.